Isolation and characterization of human monoclonal antibodies (HuMAbs) against HIV can be an important tool for illuminating aspects of the human humoral immune response to HIV that are important to vaccine development and cannot be gained by study of rodent mAbs or heterogeneous human serum Abs. Furthermore, HuMAbs against HIV may be developed as prophylactic or therapeutic reagents because of their low to negligible immunogenicity in humans. We have isolated seven HuMAbs against HIV env with interesting biological properties; these include five HuMAbs with potent neutralizing activity directed against either the CD4 binding site or V3 loop of gp120. An exciting new discovery is that two of our HuMAbs, one against the CD4 binding site (1125H) and one against the V3 loop (4117C), synergize to neutralize HIV. Preliminary results indicate that the anti-V3 HuMAb may be enhancing the ability of the anti-CD4 binding site HuMab to bind to gp120. We propose to determine the frequency with which neutralization escape mutants are selected by HuMAbs against the V3 loop and CD4 binding site and combinations of such HuMAbs, including determination of isotype, viral strain specificity, epitope specificity, and ability to: react with live versus fixed HIV-infected cells, neutralize different strains of HIV, mediate Ab-dependent, complement-mediated virolysis, and enhance infection of cells with different HIV strains. In order to facilitate production of HuMabs for a variety of studies, we propose to increase HuMAb production by optimizing the conditions for growth of HuMAb-producing cells in bioreactors, fusing our monoclonal EBV-transformed lines to human/mouse heteromyeloma, and cloning the expressed V region genes from our human monoclonal lines followed by expression of these genes as part of whole HuMAbs in mouse myeloma cells. The latter approach may be used to "rescue" the V regions from human cell lines with poor growth and/or Ab production levels, but is also important to pursue in the case of cell lines with potent anti-viral activities, such as 1125H and 4117C. The V gene usage and sequence of these HuMAbs is of interest in terms of idiotype expression and molecular modeling of the antigen binding site. In experiments extending beyond the scope of the present application, having the V regions cloned will allow us to do genetic engineering in order to produce HuMAbs with optimal anti-viral activities. Finally, in order to identify other HIV epitopes that may elicit neutralizing Abs and/or to obtain a panel of HuMAbs of different idiotypes against a given neutralizing epitope (e.g. V3), we shall isolate additional HuMAbs against HIV env and/or p18 and characterize them as described above.